• Mycobiology
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• v.50 no.2
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• pp.142-149
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• 2022

Orchids live with mycorrhizal fungi in mutualism. This symbiotic relationship plays an essential role in the overall life cycle of orchids from germination, growth, settlement, and reproduction. Among the 1000 species of the orchid, the Korean lady's slipper, Cypripedium japonicum, is known as an endangered species. Currently, only five natural habitats of the Korean lady's slipper remain in South Korea, and the population of Korean lady's slipper in their natural habitat is not increasing. To prevent extinction, this study was designed to understand the fungal community interacting in the rhizosphere of the Korean lady's slipper living in the native and artificial habitats. In-depth analyses were performed to discover the vital mycorrhizal fungi contributing to habitat expansion and cultivation of the endangered orchid species. Our results suggested that Lycoperdon nigrescens contributed most to the increase in natural habitats and Russula violeipes as a characteristic of successful cultivation. And the fungi that helped L. nigrescens and R. violeipes to fit into the rhizosphere community in Korean lady's slipper native place were Paraboeremia selaginellae and Metarhizium anisopliae, respectively. The findings will contribute to restoring and maintaining the endangered orchid population in natural habitats.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.53-53
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• 2019

Cypripediums, popularly called lady's slippers or moccasin flowers, are the showiest and most sought after hardy terrestrial orchids, collected and grown by orchid and alpine plant enthusiasts alike. In Korea, 4 species of cypripedium are reported as Cypripedium japonicum, C. macranthos, C. guttatum, and C. calceolus. We had already reported the feasibilities of C. macranthos and C. guttatum with in vitro germination methods from immature seeds. The seeds of white lady's slipper orchid (Cypripedium macranthos Sw. alba) were collected 65 days after pollination in 2018. The green pods were sterilized with flame and sowed immediately on the POM(Phytomax orchid maintenance media(R), Sigma) supplemented with BAP 0, 0.5, 1.0 mg/L and NAA 0, 1, 2mg/L. The germination of seed was observed 90 days after sowing, and the plantlets were subcultured to the same media according to the size of the protocorm with 1~2, 2~3, 3~4, 5~6, 7~8mm. The time of the subculture to the new media seems to be critical factors of forming rhizoids which is the hairy root of the cypripediums. As a results, the protorms of the white lady's slipper orchid was successfully germinated in the POM media supplemented BAP 0.5 and NAA 1.0 mg/L. The roots and rhizoids were formed in 5~6mm protocorms subculture over 95% survival ratio. We also tried to subculture to liquid medium without activated charcoal, however the browning or malformation of the roots was observed in the root. The formation of shoots from the protocorm was effectively enhanced in the POM media with non-additives of plant growth regulators. These results indicate the possibility of high and stable production and practical industrialization of endangered white lady's slipper orchids.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.10a
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• pp.37-37
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• 2018

We use two different methods for laboratory propagation from seed of lady's slipper orchid(Cypripedium macranthos Sw.); immature seed which also called green capsule or fully mature seed about 120~130 days from pollination. In green capsule culture, the seed pods should be collected within precisely right time. The right time of seed collection could be diverse under the wether conditions or nutritional factors of the plants. In fully matured seed culture, the more complicated procedures are needed to break the dormancy of the seed; thermal or chemical treatment. The seedlings in this study were easily germinated from immature seeds in Harvais medium; 53 days after pollination(DAP) in Cypripedium pubescens, DAP 65 in C. parviflorum and C. macranthos. The germinated seedlings were transplanted to hormone free media immediately to avoid abnormal growth of seedlings. When the seedlings have roots with a minimum length of around 2-3cm and have visible dormant buds, the seedlings were removed from the flask and stored in refrigerator for vernalization. To examine the correlation of seedlings and maternal plants, the 125 seedlings of C. macranthos were transplanted in the soil bed at a distance of 20-100 cm from mother plants on April 20. The survival rate of seedlings were 92% in 20 cm distance from the ripe plants, and 56 % in 100 cm distance. The seedlings which were transplanted near mother plants showed vigorous growth in plant height, leaf width, and especially dormant buds. Considering the existence of mycorrhiza which is a symbiotic association between a fungus and the roots of a orchid vascular, the various fungus from mother plants could affect the growth of the seedlings. These results indicate the possibility of high and stable production and practical industrialization of endangered lady's slipper orchids.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.45-45
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• 2018

We had already reported the successful germination for green pods of purple lady's slipper orchid (Cypripedium macranthos Sw.). The green pod methods is to take immature seeds in green capsules, sterilize the capsule, and take out the sterile seeds. This method, however, needs very critical time of harvest. The critical time of seed harvest changes depending upon the species, condition of the specimen, and climatic influence, and the right time lies between 5 and 12 weeks after fertilization. In this study, the mature seeds were collected after 120-130 days with hand-polination of lady's slipper orchids. Mature seeds are usually dormant and it has to be overcome, either with hormone or storing the seeds near freezing for two or three months to break dormancy. The seeds were first surface sterilized with 70% ethanol and then transferred 1% NaOCl for 10-15 minutes, followed by rinses 3 times with sterilized distilled water. The cypripedium seeds consists of an embryo within a seed coat known as a testa. The testa is water repellent and the seed has a large air space between the embryo and testa so the seed tends to float on water. We had resolved the problems with vacuum pump to soak water into the testa before sterilization. The seeds were placed on liquid or agar solidified germination media. Cultures were incubated at $24{\pm}1^{\circ}C$ in dark. The seeds were germinated in 6-8 weeks in liquid suspension culture (germination percentage over 18%); however, the seeds on agar solidified media took more than 5 months to germinate and the germination percentage less than 5%. The most effective media for liquid culture was 1/4 strength Murashige and Skoog (MS) medium with 50 ml/l coconut water ($4brix^{\circ}$) at pH 5.8.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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• pp.23-23
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• 2019

Cypripedium is an orchid genus of over 50 species widely distributed in the northern temperate zone, with a few taxa extending central America and the Himalayas and the southwesternmost tip of China in Asia. Of the known species approximately two thirds are found in China. In Korea, 4 species were reported to find with rare popularity ; C. macranthos, C. guttatum, C. japonicum, and C. calceolus. The seed has a firm brown fusiform testa with an opening at one end where the micropyle of the ovule was situated. Cypripedium has small, fusiform, wind-dispersed seeds that weigh about $1{\sim}2{\mu}g$ and range from 0.1 to 2 mm long and 0.07 to 0.4 mm across. The germination of Cypripedium was previously thought to be hard to cultivate in one's garden and impossible to propagate artificially, nowadays lab-propagated seedlings are readily available in EU or United States. We had already reported the successful micropropagation of lady's slipper orchids with green pod sowing methods. The collected Cypripedium species and hybrids were planted in Korea National Arboretum (KNA) on Oct. 2018. The 11 species including C. candidum and the 31 hybrids such as C. Ingrid were introduced from Germany with CITES certification. The lady's slipper orchids bloomed on May to June, and the flowers were pollinated in the same species or hybrids to get seeds for in vitro germination on 2~3 days after fully flowering. The green pods were collected after 49~70 days after pollination. The 6 species of Cypripedium seeds were easily germinated on the POM within 70 days after sowing, and the 12 hybrids PLBs were observed with small roots. We had established the scheme of lady's slipper orchids germination in 3 months with green pods, and get the possibility of seedling in 10 months of sowing in vitro.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.138-145
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• 2016

To find the optimal propagation condition for endangered Cypripedium macranthos Sw., also known as lady's slipper orchid, the effect of various organic additives on in vitro germination, protocorm formation and seedling growth was investigated during asymbiotic seed culture. When $100ml{\cdot}L^{-1}$ coconut water was added to the basal medium, the highest germination rate and protocorm formation rate were achieved, with 70.8% and 74.2% respectively. Supplementation of phloem sap from birch tree or maple tree also showed a facilitating effect to improve the germination and protocorm development. With $100ml{\cdot}L^{-1}$ birch sap or maple sap, both the germination and protocorm formation rates were roughly more than 65% and 68%. The roots and buds of the seedlings grew vigorously in the medium containing $100ml{\cdot}L^{-1}$ coconut water or phloem sap, in particular, their bud formation rates increased by more than 70%. Addition of banana powder and peptone could not create a more significantly favorable culture condition, and non-addition had the worst results. Our results demonstrated that proper organic amendments such as coconut water and phloem sap might be preferred to in vitro germination and the growth of seedlings developed from the protocorm of C. macranthos Sw. during asymbiotic seed culture.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.132-137
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• 2016

The genus Cypripedium, known as lady's slipper orchid, has been considered to have a considerable marketability as a potted and garden plant with beautiful flowers; however, this species is becoming endangered and, in some places extinct, due to habitat destruction and illegal collections. As such, an optimized artificial propagation system is necessary for its conservation and horticultural cultivation. For the establishment of the in vitro proliferation of the endangered Cypripedium macranthos Sw., native to Korea, the effects of medium strength and sucrose concentrations on germination, protocorm formation and seedling growth were investigated through asymbiotic seed culture. The highest germination rate and protocorm formation rate were obtained with a 1/4 MS medium; higher MS medium strengths did not generate the favorable conditions required for germination and protocorm growth. The overall growth characteristics of roots and buds developed from protocorms were best in cultures of 1/4 MS medium. On this medium containing $10g{\cdot}L^{-1}$ sucrose, both the germination rate and protocorm formation rate increased significantly. The general growth properties of seedlings developed on the medium with $10g{\cdot}L^{-1}$ sucrose were best, showing the highest bud formation rate and root number. Our results demonstrate that the 1/4 MS medium, supplemented with $10g{\cdot}L^{-1}$ sucrose, could improve in vitro germination, and facilitate the growth of seedlings developed from the protocorms of C. macranthos Sw.

• The Korean Journal of Mycology
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• v.50 no.4
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• pp.361-365
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• 2022

Here, Podospora leporina was isolated and cultured from Cypripedium guttatum Sw. (spotted lady's slipper), which was designated as an endangered wildlife class 1 by the Ministry of Environment. Further, Podospora sp., which is found in Calanthe discolor Lindl. (Japanese hardy orchid) root is recorded as an endophytic fungus in Jeju Island, Korea. However, there is no existing record of Podospora spp. Thus, in this study, we analyzed the morphological characteristics and two molecular markers, internal transcribed spacer (ITS) and large ribosomal subunit (LSU), of P. leporina and compared these data with other relative species. Consequently, this endophytic fungal species was confirmed as an unrecorded species in Korea, so we presented cultural characteristics and microstructures, such as the conidia of P. leporina.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2020.08a
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• pp.11-11
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• 2020

Background : The genus Cypripediums are typically adapted to the low temperature and cold climates of the high land in Korea. The species which were reported in Korean peninsula are C.japonicum, C.macranthos, C.calceolus and C.guttatum. We had already reported the successful germination of the C.macranthos and C.guttatum. The small spotted lady's slipper orchid(C.guttatum) is most endangered plants to extinction of which population was reported less than 100 in Korea. The objective of the present research is restoration of the C.guttatum in ex situ and adaptation in high land and nursery. Methods and Results : The 350 seedlings of C.guttatum were transplanted in soil at alpine area(altitude 750m) and the nursery of Korea National Arboretum(KNA) in YangPyeong city on May 2019 and 2020 in order to verify the possibility of ex situ restoration. The sprouted ratio of the seedlings were less than 10% in the alpine area and 83% in the nursery of KNA. The reasons of the diverse survival ratio are presumed as the low moisture content in the soil and heavy shade of the alpine area. The 15 cypripedium species and 29 hybrids were also effectively adapted in the KNA rare plant garden. Conclusion : These results demonstrated the difficulties of the restoration for small spotted lady's slipper orchid in the natural habitation. From the above results, we may conclude that the critical factors of the genus Cypripedium are intensive care for the fragile seedlings during the 1^st year acclimatization.